Anti-idiotypic nanobody_ A strategy for development

Talanta 143 (2015) 388–393

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Talanta journal homepage: www.elsevier.com/locate/talanta

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Anti-idiotypic nanobody: A strategy for development of sensitive and green immunoassay for Fumonisin B1 Mei Shu a,b, Yang Xu a,b,n, Dan Wang c, Xing Liu a,b, Yanping Li b, Qinghua He b, Zhui Tu a, Yulou Qiu a,b, Yanwei Ji a,b, Xianxian Wang a,b a

State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China Sino-Germany Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China c Key Lab for Agricultural Products Processing and Quality Control of Nanchang City, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, People's Republic of China b

art ic l e i nf o

a b s t r a c t

Article history: Received 20 March 2015 Received in revised form 27 April 2015 Accepted 4 May 2015 Available online 11 May 2015

Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2β Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2β Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95 7 0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27– 5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1–BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2β Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2β Nb replacing the antigen–carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens. & 2015 Elsevier B.V. All rights reserved.

Keywords: Anti-idiotypic antibodies Nanobodies Phage display library

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals and reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biopanning and identification of Ab2β Nb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Expression and purification of the Nb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Quantitative immunoassay established with Nb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Affinity measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Result and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Selection and sequence analysis of FB1-specific Nb phages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Nb-ELISA for FB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Optimizing the competitive ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Kinetics analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Assay validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

389 389 389 389 389 390 390 390 390 391 391 391 392

n Corresponding author at: Sino-Germany Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China. Tel.: þ 86 791 88329479; fax: þ 86 791 88333708. E-mail address: [email protected] (Y. Xu).

http://dx.doi.org/10.1016/j.talanta.2015.05.010 0039-9140/& 2015 Elsevier B.V. All rights reserved.

M. Shu et al. / Talanta 143 (2015) 388–393

389

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

1. Introduction Fumonisins are a group of environmental toxins, mainly produced by Fusarium species and Gibberella fujikuroi that grow on agricultural commodities in the field or during storage [1,2]. More than 10 types of fumonisin structures have been identified and characterized. Among these, the B-series has been confirmed as the major type of fumonisin produced in nature, with fumonisin B1 (FB1) being the most abundant and toxic [3]. FB1 is possibly associated with human cancer and has been classified by the International Agency for Research on Cancer as a possible group 2B human carcinogen [4]. Rapid and sensitive immunoassays have been developed for FB1 to minimize the risk of FB1 to humans and animals. These immunoassays include ELISA [5], immunomagnetic beads ELISA [6], fluorescent immunosorbent assay [7], and microarray assay [8]. However, FB1 is a hapten and is not sufficiently large for direct detection. It has to be conjugated to carrier proteins, such as ovalbumin, bovine serum albumin, and keyhole limpet hemocyanin, as competing antigens to enhance sensitivity [9]. The synthesis of a hapten–carrier is time consuming, expensive, and may pose a threat to manufacturers' health. Moreover, the antibody concentration must be optimized to ensure optimum determination of the hapten using different batches of hapten conjugates [10]. To develop a reliable and green detection immunoassay, researchers focused on searching for an easy and environment-friendly method to obtain hapten substitutes. They found that peptides and proteins can substitute the synthetic antigen and bind site to the antibody [11]. Peptides (termed mimotopes) can be obtained by phage display [12–14] and self-assembly [15,16]. Nevertheless, the small peptide molecule must be linked to the phage particles or expressed as a fusion protein [17]. Meanwhile, producing a selfassembling peptide that limits its real applications in mycotoxin immunoassays is very difficult and expensive. Generating anti-idiotypic antibodies (Ab2) is another alternative approach to surrogate antigens. Ab2s are the second antibodies, which can compete with external antigens in binding the same variable region of specific antibodies [18]. This antibody can be classified into four distinct categories (α, β, γ, and ε), and only the Ab2β internal image antibodies can bind to the antigen-combining site and resemble the original antigen [19]. Some antiidiotypic antibodies effectively mimic the biological functions of the hapten. As early as the 1990s, Ab2s for large molecules have been well developed and used for various applications in therapy and immunoassays [20]. Polyclonal and monoclonal techniques have successfully generated numerous anti-idiotypic antibodies against small molecular weight haptens, including insecticides, herbicides, hormones, and mycotoxins [21–25]. Nevertheless, the recognition surfaces of the polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) are large flat areas, which seriously limit their use for mimicry of small haptens [10]. Recent success in generating Ab2 by genetic engineering techniques, such as single-chain variable fragments [26], mAbs [27], and nanobodies (Nbs) [11], prompted our interest in generating an Ab2β Nb that is specific for anti-FB1 mAb through phage display technology. In this study, the Ab2β Nb as a coating antigen was isolated from a naïve phage library, leading to the development of an immunoassay for FB1 with more sensitivity to that produced by the hapten-conjugates. Excellent recoveries were found in corn,

rice, and feedstuff samples. The Ab2β Nb as a hapten substitute is an important contribution to the immunoassay technology for small molecules.

2. Materials and methods 2.1. Chemicals and reagents Fumonisin B1, Fumonisin B2, Deoxynivalenol, Ochratoxin A, Zearalenone and Aflatoxin B1 were purchased from Sigma (St. Louis, MO, USA). Nco I, Not I, T4 DNA ligase, Cycle-Pure Kit, and Gel Extraction Kit were purchased from Takara (Dalian, China), and 1 mL His Trap™ HP and HRP-conjugated anti-M13 antibody were obtained from GE Healthcare Inc. (Piscataway, NJ, USA). The commercial ELISA kit was provided by Shenzhen Lvshiyuan Biotechnology (Lvshiyuan, China). Naïve phage libraries, anti-FB1 mAb, and FB1–BSA conjugates were prepared in our laboratory [17]. 2.2. Biopanning and identification of Ab2β Nb Ab2β Nbs against anti-FB1 mAb were isolated from the naïve Nb phage display library constructed by our research team in our laboratory [28]. In brief, in the first round of panning, 100 μL of the naïve Nb phage display library (1012 cfu/mL) was added to a 100 μg/mL anti-FB1 mAb coated well and incubated at 37 °C for 60 min. After washing with PBST [0.01 M PBS (pH 7.4)þ0.1% (v/v %) Tween-20], 100 μL of elution buffer [0.2 M glycine–HCl (pH 2.2), 1 mg/mL BSA] was added and gently shaken for 10 min at room temperature. The eluate was pipetted into a microcentrifuge tube and neutralized with 15 μL of 1 M Tris–HCl. For the second and third panning, the plate was coated with anti-FB1 mAb at 25 and 10 μg/ well, respectively, and the concentrations of free FB1 for the elution of bound phage nanobodies were 10 and 5 ng/mL, respectively. The positive colonies were subjected to competitive phage ELISA. Preparation, incubation, and washing of plates were conducted as described above. The positive phage particles were mixed with an equal volume of analytical standard FB1 in the plates. After incubation and washing, the phages bound to anti-FB1 mAb were detected as previously described. The clones that were inhibited from binding when FB1 was present in the solution were sent to Invitrogen (Shanghai, China) for sequencing. 2.3. Expression and purification of the Nb Fig. 1A shows the schematic of constructing expression plasmid. The expression vector pET-B26 was constructed by cloning the special DNA, which encodes the Nb fragment into the vector pET25b. The pET-B26 was then transformed into Escherichia coli BL21 (DE3) cells, and the cells were grown in 100 mL of LB media containing ampicillin (100 mg/mL) at 37 °C until the OD600 reached approximately 0.6. Subsequently, the target proteins were produced in an auto-induction media at 30 °C by shaking at 250 rpm for 12 h [29]. The cells were harvested by centrifugation at 1000 g for 10 min. The harvested cells were washed with 20 mL of 10 mmol/L Tris–HCl (pH 7.4) and resuspended in 10 mL of 50 mmol/L Tris–HCl (pH 8.0). The resuspended cells were broken by ultrasonic cell disruption, and the crude cell lysate was obtained by centrifugation at 8000 g for

390


Fig. 1. (A) Schematic of the construction of the expression plasmid for the Nb protein. (B) Competitive phage ELISA based on B26, soluble Nb and FB1–BSA conjugates. Data are represented as an average7standard deviation of three replicates. (C) Amino acid sequence of B26 Nb from third round of panning. Recombinant Nb CDR (red) and FR delimitations from the IMGT V domain directory. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

10 min. The target proteins with 6 His tags were purified using NiNTA affinity columns and analyzed by 12% SDS-PAGE.

2.4. Quantitative immunoassay established with Nb As for the assay, 100 μL of Nb fusion protein (diluted in PBS, pH¼7.4) was added into the 96-well microplate (Costar, No. 42592) and incubated at 4 °C overnight. After blocking with 5% skimmed milk–PBS (300 μL/well) and washing with PBST, 50 μL of serial concentrations of FB1 (from 0.1 ng/mL to 25 ng/mL diluted in PBS) or sample extracts equal with anti-FB1 mAb were added to the wells, and the mixture was incubated at 37 °C for 30 min. Followed by washing three times with PBST, 100 μL of 1/2000 dilution of HRPconjugated goat anti-mouse antibody was incubated in the wells at 37 °C for 30 min. Finally, 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate was added to the washed wells. The optical density was detected at 450 nm on a microplate reader (Thermo Scientific, USA). To determine the optimized dilution of the immunoassay reagents, a check-board assay was conducted using different dilutions of fusion proteins and anti-FB1 mAb in advance.

2.5. Affinity measurements Affinity sensors were employed to measure the interaction of immobilized Ab2β Nb and anti-FB1 mAb by using a label-free biosensor system (Pall Corporation, USA). Ni-NTA biosensors specifically bound to His-tag attached to 20 μM of the Nb. A dilution series of at least four concentrations of anti-FB1 mAb (12.5, 25, 50, 75, and 150 nM) were injected into the cuvette. The obtained data were analyzed using the BLItz software with the association phase, resulting in the association and dissociation rate constants. To compare the phage performance as a recognition reagent in the biosensor, we also immobilized FB1–BSA conjugates on Amine Reactive 2nd Generation biosensor and analyzed as previously described [30].

3. Result and discussion 3.1. Selection and sequence analysis of FB1-specific Nb phages Three rounds of panning were performed to isolate FB1-specific Nb phages from a naïve phage Nb library, whose size was 1.9 107 cfu/mL


indicated that the Nb library satisfied with high sequence diversity [28]. The bound phages were eluted using glycine–HCl (pH 2.2) in each cycle of panning to obtain Nb phages with increasing sensitivity to FB1. Titers of output phage increased after each round. The quantity of elutant in the third panning was approximately 10 orders of magnitude more than the first cycle, indicating the effective enrichment of specific clones binding to FB1. To isolate FB1-specific Nb phages, we randomly selected 96 clones from each round of panning. Indirect competitive phage ELISA against anti-FB1 mAb was performed to identify the individual clones. Only clone B26 from the third round showed specific binding to anti-FB1 mAb, which can be inhibited by FB1 in competitive ELISA with the IC50 of 1.0770.2 ng/mL (Fig. 1B). The main feature of the nanobodies is the occurrence of amino acids, F(Phe), E(Glu), R(Arg), and F/G(Gly) in the FR2, which stabilizes the Nb fragments and the extended length of CDR3 regions [31]. The amino acid sequences of the Ab2β Nb in the region agree well with the findings (Fig. 1C). Clone B26 has F, R, and G in FR2 and has a longer CDR3 region with 21 amino acid residues without significant amino acid variation. The DNA sequence alignment indicated that B26 belonged to lama glama (llama; IMGT/DomainGapAliga). 3.2. Nb-ELISA for FB1 The Nb fragment from B26 clone was subcloned into pET25b vector. The Ab2β Nb with a 6 His tag was harvested by purifying cell lysates through the Ni-NTA affinity columns. The molecular mass of the purified Nb was determined on a 12% SDS PAGE gel as 17 kDa major bands (Fig. 1B). A competitive immunoassay for FB1 was performed to optimize the concentrations of the Ab2β Nb and anti-FB1 mAb in advance. The sensitivity of the assay was determined. The limit of detection of the immunoassay established with the Ab2β Nb, estimating from the mean (plus two standard deviations) of the 10 blank samples was 0.15 ng/mL, and the IC50 of the assay was 0.957 0.12 ng/mL (Fig. 1B). The linear range calculated by 20–80% inhibition of Nb-ELISA was 0.27–5.92 ng/mL. The sensitivity of Nb-ELISA was improved 20 times than that of FB1–BSA based ELISA (IC50 ¼21.14 71.05 ng/mL) under the optimal conditions [32]. Wang et al. [11] reported that Nb-ELISA exhibits a lower sensitivity (IC50 ¼13.8 μg/kg) compared with the conventional ELISA (IC50 ¼1.2 μg/kg). The results of the present study revealed that the Ab2β Nb was more appropriate as a coating antigen for FB1 analysis than the hapten-conjugates, which requires complicated synthesis procedures and are produced at a small scale. The specificity of the assay was tested, but negligible cross reactivity was observed with AFB1, ZEN, DON, and OTA, except for FB2 which exhibited 4.93% cross reactivity (Fig. 2).

Fig. 2. Cross reactivity of Nb-ELISA with common mycotoxins. Data are represented as an average7standard deviation of three replicates.

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3.3. Optimizing the competitive ELISA Immuno-reactions can be seriously influenced by ionic strength and pH [33]. Thus, these two parameters were investigated to evaluate their effects on the interaction between anti-FB1 mAb and the Ab2β Nb. Different concentrations of buffer ionic strength (from 5 to 50 mM) exhibited evident impact on the established competitive curve (Fig. 3A). The obtained results indicated that the value of maximum signal decreased when the ionic strengths were 0, 25, and 50 mM. The lowest IC50 value was obtained at a concentration of 10 mM. The influence of the assay buffer with different pH values was also examined at 5.0–9.0 (Fig. 3B). The IC50 values had no significant differences among standard curves at pH 5.0–8.0, whereas the IC50 of assay turned straight and the optical signal markedly decreased at pH 9.0. The pH 7.4 (IC50 ¼ 0.9670.09 ng/mL) was used as the optimum pH condition for the assay based on the favorable IC50 value. The best performance was achieved at 10 mM PBS in pH 7.4. These results resemble with the expression of Nbs or peptide fusion protein of some hapten-type toxins [11,17]. 3.4. Kinetics analysis Affinity measurement of the purified Nb fragment was developed on a label-free biosensor. The Ab2β Nb was immobilized on the biosensor surface, and the antibody remained in an unbound state in solution. Original data were collected and processed using the BLItz software. The sensorgram curves were fit to a 1:1 interaction model (AþB¼AB). The equilibrium dissociation constant (KD) measured for Ab2β Nb: anti-FB1 mAb was 164.6 nM, which was 270 times lower

Fig. 3. Effects of ionic strength (A) and pH (B) on the performance of Nb-ELISA. Data are represented as an average7standard deviation of three replicates.

392


Table 1 Recoveries of FB1 added to samples in determinations performed by Nb-ELISA. Sample type FB1 added (mg/ kg)

Corn

Rice

Feedstuff

10 50 100 500 1000 10 50 100 500 1000 10 50 100 500 1000

Nb-ELISA (n¼ 3) (mg/kg) Average recovery (%) 7.92 7 0.52 48.527 3.14 103.767 3.28 558.617 6.01 1121.53 7 15.44 7.56 7 0.41 43.22 7 3.98 105.58 7 8.54 539.727 9.11 1072.357 17.36 7.197 0.52 42.29 7 2.57 95.667 4.88 546.84 7 7.31 1100.43 7 10.78

79.20 97.04 103.76 111.72 112.15 75.60 86.44 105.58 107.94 107.24 71.90 84.58 95.66 109.37 110.43

Table 2 Detection results of incurred samples by Nb-ELISA and the commercial ELISA kit.

Fig. 4. (A) Kinetic analysis of the interaction between Nb and anti-FB1 mAb by NAT biosensor. (B) Kinetic analysis of the interaction between FB1–BSA and anti-FB1 mAb by amine biosensor.

than that measured for FB1–BSA:anti-FB1 mAb (KD ¼0.62 nM; Fig. 4). In general, nanobodies isolated from naïve phage display libraries were not suitable as antibodies because of their low micromolar range. Nevertheless, the Ab2β Nb as antigen and small analytes, such as mycotoxins, that can be determined by a competitive immunoassay are special. According to the principle of indirect competitive immunoassay [34,35], weaker affinity indicates less amount of analyte needed to participate in the competition; therefore, the sensitivity is improved. We inferred that the Ab2β Nb was more fit for working as a coating antigen than the hapten-conjugates because of the lower affinity of Ab2β Nb to anti-FB1 mAb.

Samples

Nb-ELISA (n ¼3)a (mg/kg)

Commercial ELISA kit (n¼ 3)a (mg/kg)

Corn C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12

158.9 7 4.2 200.17 8.9 341.8 7 7.8 162.7 7 3.3 71.6 7 4.4 2322.7 7 17.2 143.0 7 9.5 576.9 7 5.4 708.6 716.5 210.7 76.2 149.8 710.1 15.5 7 2.7

165.3 7 5.1 199.2 7 12.6 301.5 7 9.8 172.7 7 9.5 59.9 73.7 1986.4 7 25.8 185.7 7 10.2 596.2 711.7 716.8 720.4 198.1 7 5.6 160.4 7 11.2 NDb

Wheat W1 W2 W3 W4 W5 W6 W7 W8 W9 W10

20.7 7 4.3 20.4 7 2.6 26.9 7 7.7 ND ND 1259.0 7 33.8 17.2 7 5.0 8.3 7 1.0 ND 41.3 7 3.6

27.5 7 5.8 ND 35.87 8.2 ND ND 1246.6 7 35.6 ND ND ND 40.8 73.8

Rice R1 R2 R3 R4 R5

ND ND ND ND ND

ND ND ND ND ND

3.5. Assay validation a

Spike and recovery analysis with Nb-ELISA was conducted with corn, rice, and feedstuff samples to evaluate effectiveness of the assay for FB1 analysis. These samples were spiked with known concentrations of FB1 (10–1000 μg/kg). Recovery rates from 71.90% to 112.15% are shown (Table 1). The Nb-ELISA and the commercial ELISA kit were used to detect 27 cereal samples purchased from markets in China. Results of Nb-ELISA showed that 19 samples were contaminated with FB1, whereas only 15 samples were positive by commercial ELISA kit (Table 2). The correlation (R2 ¼0.992) was acquired between Nb-ELISA and commercial ELISA kit. Dilution of the extract is an effective approach to eliminate matrix effect. Sample extracts were diluted 20-, 200-, and 2000-fold and were used to prepare serial concentrations of

b

Each assay was carried out in three replicates on the same day. Not detectable.

FB1 standards for Nb-ELISA. Taking the dilution into account, the linear range was 4.91–1468.4 mg/kg which is suitable for monitoring total fumonisin concentration under the current regulatory limits of fumonisins in most countries.

4. Conclusions In this work, one type of Ab2β Nb against FB1 was isolated from a naïve alpaca Nb phage display library. This antibody was subjected


to develop a sensitive and environment-friendly immunoassay. Ab2β Nb is a novel immunochemical reagent suitable for various purposes in FB1 analysis and other small molecular weight compound works. Furthermore, other advantages of using the Ab2β Nb approach for mycotoxin analysis include its simple and low-cost production of homogeneous and non-toxin products. The sensitivity of the Nb-based immunoassay was improved approximately 20-fold than that of the hapten-conjugates. These results indicate that Ab2β Nbs will be an excellent tool for various kinds of small molecule compound immunoassays in the future.

Acknowledgments This work was financially supported by the National Basic Research Program of China (Grant no. 2013CB127804), the National Natural Science Foundation of China (Grant nos. NSFC-31471648, NSFC-31360386, NSFC-31201360, and NSFC-31171696), the Jiangxi Province Key Technology R & D Program (Grant no. 2014BBG70090), the Natural Science Foundation of Jiangxi, China (Grant nos. 20132BAB214005 and 20143ACB21008), and the Education Department of Jiangxi Province (Grant no. GJJ13095).

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